A recurrent synonymous L1CAM variant in a fetus with hydrocephalus

We report the case of a hydrocephalic fetus in which clinical exome sequencing revealed a recurrent synonymous variant of unknown significance, c.453G>T, in the L1CAM gene. This report presents the second case of X-linked hydrocephalus in a fetus with this variant. Since we reproduced the RNA analysis, we were able to reclassify this variant as likely pathogenic. Our results stress the importance of not excluding synonymous variants during prioritization.

After genetic counseling and receiving written informed consent from the couple, we performed clinical exome sequencing (CES) followed by trio analysis to determine the etiology of the hydrocephalus.Genomic DNA was extracted from the peripheral blood of the couple (Fig. 1b, II.1 and II.2) and from the aborted fetal tissue (Fig. 1b, III.1) using a standard protocol.A clinical exome library was generated using SOPHiA Clinical Exome Solution v2 (Sophia Genetics) following the manufacturer's protocol.The library was sequenced on a MiSeq (Illumina, San Diego, CA, USA) in 300 bp paired-end mode.Sequence reads were aligned to the UCSC human reference genome (GRCh37/hg19 assembly).Our in-house pipelines included FastQC v0.11.8, Bowtie2 v.2.3.5, Picard v2.21.6, SAMtools 1.10, Freebayes v1.3.1, and bedtools v2.29.2.The Variant Call Format file was uploaded to the Franklin Analysis platform (Genoox) for variant annotation, classification, and filtration.The following filter criteria were used: (1) minor allele frequency <0.05 in both the general population (aggregated frequency) and the in-house database (internal frequency); (2) associated with the HPO term hydrocephalus (HP:0000238); (3) excluded benign, likely benign and VUS-leaning benign Franklin classification; (4) >10 coverage; (5) >100 quality; and (6) excluded benign prediction (aggregated prediction).We obtained a final list of six variants.Considering the disease inheritance model and zygosity of the variant, only a hemizygous synonymous variant of unknown significance in L1CAM was retained (Fig. 1c).Despite its classification as a variant of unknown significance, this variant has already been described as the cause of HYCX 5 .
Using Sanger sequencing, we confirmed that this variant was present in the hemizygous state in the fetus (III.1) and in the heterozygous state in the mother (II.2) (Fig. 1d).Additionally, Sanger sequencing of available members of the family (Fig. 1d) did not reveal a heterozygous variant in the mother's parents (I.1 and I.2), indicating de novo occurrence of this variant.Prenatal testing was conducted during the second pregnancy, and the variant was not detected in a female fetus unaffected by hydrocephalus (III.2) (Fig. 1d).
According to previous report 5 , the synonymous variant creates a novel 5' splice site, resulting in the deletion of part of the exon.We decided to perform an independent evaluation of variantinduced splicing alterations.Since we were unable to extract RNA from the fetus (III.1),we extracted RNA from the peripheral blood of the proband's mother (II.2) and retrotranscribed it into cDNA.RT-PCR and Sanger sequencing of cDNA were carried out to amplify exons 4-5 in L1CAM mRNA using forward (CTCAGAGGTTC-CAGGGCATC) and reverse (TCGTCCTGCTTGATGTGCAA) primers.Agarose gel electrophoresis of cDNA products revealed not only full-length (longer) product but also aberrantly spliced (shorter) product that was not detected in the healthy control (Fig. 2a).Sanger sequencing of the cDNA products confirmed that the variant created a novel strong 5' splice site, thus leading to an inframe 72 bp deletion at the end of exon 5 (Fig. 2b, c).Consequently, 24 amino acids in immunoglobulin-like (Ig-like) domain 2 were lost.
During analysis, the variant was classified according to ACMG-AMP variant interpretation guidelines 7 as a variant of unknown significance in Franklin (PM2) and as a likely benign variant in Varsome (PP5, PM2, BP4, BP7).We provided evidence of pathogenicity through a functional study to identify the splicing effect of the variant on RNA (PS3, PM4) and by identifying another unrelated fetus with the same variant exhibiting the same clinical features of the disease.According to our findings, we reclassified the variant as likely pathogenic.
Synonymous variants are often overlooked in NGS analysis.Since silent variants are usually nonpathogenic, they are excluded during filtering.For example, the default filter setting in Franklin excludes synonymous variants.Our study showed that synonymous variants should not be filtered out by default during NGS analysis.Re-evaluation of synonymous variants may increase the diagnostic yield of inherited disorders.Moreover, splicing variants resulting in in-frame deletions tend to be evaluated as variants of unknown significance.Therefore, reporting symptomatic patients harboring these variants is highly important for their clinical classification.
In conclusion, we report the recurrence of a hemizygous L1CAM variant, which is responsible for hydrocephalus in male fetuses.Since pathogenic synonymous variants in L1CAM are extremely rare, we provide crucial clinical evidence that synonymous variants should not be filtered out during prioritization steps.

HGV DATABASE
The relevant data from this Data Report are hosted at the Human Genome Variation Database at https://doi.org/10.6084/m9.figshare.hgv.3360.

Fig. 2
Fig. 2 RNA analysis.a Agarose gel electrophoresis of cDNA products showing different-sized products in the heterozygous mother (II.2) in contrast to the healthy control (WT).b Illustration of variant-induced splicing resulting in a 72 bp deletion.c cDNA sequence electrophoretograms of L1CAM.In II.2, forward-direction sequencing revealed the mixed sequence immediately before the novel splice site in exon 5, and reverse-direction sequencing was performed before the beginning of exon 6.